Analytical study of molecular interactions requires the ability to observe changing molecular concentrations in an environment of interest, and also the ability to quantify chemical kinetics and affinity energetics. This is particularly true in biological contexts, where processes at the cellular and even organismic level often can be traced to changing concentrations and/or binding energetics of biomolecules and cofactors. For example, proteins and their byproducts play a critical role in nearly every event that take place within living cells. The ability to characterize these events is furthered by the ability to profile the concentration of specific proteins as a function of time and various physiological conditions, and the ability to measure the affinity and energetics of protein binding reactions. The rate at which these parameters are measured by current methodology, however, is often limited by requirements for large sample volumes and time-consuming modifications of the molecules under investigation.
For example, relative biomolecular concentration is frequently assayed using two-dimensional gel electrophoresis, a procedure that is time-consuming, labor-intensive, and which requires significant technical expertise to obtain quantitative information. One approach toward circumventing these limitations is to develop the equivalent of a DNA microarray for biomolecules such as proteins. A representative system utilizing this approach involves an antibody array sensitive to the amounts and modification states of endogenous ErbB receptor tyrosine kinases. The array has been employed to monitor the kinetics of signal transduction in human tumor cell lines in a multiplexed fashion. See, e.g., Jenkins et al., “Arrays for protein expression profiling: Towards a viable alternative to two-dimensional gel electrophoresis?”, Proteomics 1:13-29 (2001).
One requirement for protein arrays that currently limits their widespread use is the need to label sample proteins with a fluorescent marker. Although the labeling process is straightforward for DNA, the complexity of a protein's structure poses significant challenges to attaching labels to specific sites while preserving the functionality of the protein molecule. The development of label-free protein detectors that are scalable for array applications is ongoing. Although approaches such as the quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) can detect label-free binding, they are difficult to miniaturize for protein arrays. See, e.g., O'Sullivan et al., “Commercial quartz crystal microbalances—theory and applications,” Biosensors and Bioelectronics 14:663 (1999); Green et al., “Surface plasmon resonance analysis of dynamic biological interactions with biomaterials,” Biomaterials 21:1823 (2000).
One limitation of existing label-free detectors is that they tend to be significantly less sensitive than fluorescence detection. Devices such as the QCM, SPR, and microcantilever typically resolve 103 to 104 molecules per μm2, whereas confocal fluorescent microscopy can routinely resolve in the range of 1-10 molecules per μm2.
The need for large sample volumes affects not only devices for measuring concentration, but also techniques for measuring binding energetics. The latter are also limited by the difficulty of monitoring a particular species of interest in a reaction environment. Calorimetry, for example, has been applied to thermodynamic characterization of ligand binding processes. Experiments may be performed at a constant temperature by titrating the ligand into a solution containing the binding partner in the sample cell of the calorimeter; see, e.g., Jelesarov et al., “Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition,” Journal of Molecular Recognition 12:3 (1999). After each addition of the ligand, the released or absorbed heat in the sample cell is measured relative to a reference cell filled with buffer. Both the enthalpy and binding constant can be obtained by measuring the energy change as a function of the ligand concentration. The amount of ligand consumed in an experiment may be, for example, 10-100 nmol.
Conventional calorimeters exhibit the disadvantages noted above. They typically require large sample volumes—typically in the range of 0.1 to 1 ml. In many cases, yields of the product of interest are too low to be detected within such large volumes. Calorimetry is also non-specific; the heat measured is not necessarily produced by the molecules of interest. This makes it difficult to analyze signals from complex reactions unless results from specific analytical measurements are already available.
Although several researchers have shown that microfabricated devices can detect the thermal properties of sample volumes less than a nanoliter (see, e.g., Berger et al., “Thermal analysis using a micromechanical calorimeter,” Applied Physics Letters 69:40 (1996); Nakagawa et al., “Picojoule and submillisecond calorimetry with micromechanical probes,” Applied Physics Letters 73:2296 (1998); Johannessen et al., “Heat conduction nanocalorimeter with pl-scale single cell measurements,” Applied Physics Letters 80:2029 (2002)), demonstrating that microfabricated calorimeters can be highly sensitive, these devices have not yet been integrated with microfluidics. Accordingly, there remains a need for convenient analytical techniques for accurately measuring concentrations and binding energetics, particularly in connection with small sample volumes containing diverse constituents.